Etude cinétique de l'action de la trypsine sur la β-lactoglobuline native ou dénaturée par la chaleur

Abstract

The mechanism constants and the maximum rates of reaction have been compared at various temperatures between 20 and 45°C for the reaction comprising the hydrolysis of native lactoglobulin by trypsin. The comparisons have also been made for two other forms of lactoglobulin (“ s” and “ f”) denatured by heat. It was found that for the native substrate, K mN is equal to 0.8 · 10 −4 M and does not vary appreciably with the temperature. However, for the two denatured forms, the Michaelis constant (calculated with the molecular weight of the native form) is three times as small as that for the native form, and in addition is practically independent of temperature. The relative values for the rate constant of the decomposition of the enzyme-substrate complex change with the temperature. The energies of activation are respectively 18,600 cal/mol. for the hydrolysis of native lactoglobulin and 14,200 cal/mol. for that of the denatured forms. Using these data, the variation of entropy and enthalpy of activation were calculated according to the absolute rate theory and the values found were respectively ΔS ∗ = −4.5 cal/degree and ΔH ∗ = + 18,000 cal/mol. for the native substrate, ΔS ∗ = −18 cal/degree and ΔH ∗ = + 13,600 cal/mol. for the denatured substrates. The interpretations which one can give to these results taking into consideration the complexity of proteolytic reactions, are discussed. The results appear to favour the idea that the limiting process of the overall rate corresponds in the native protein to a simultaneous breaking of a peptide linkage and of three out of four hydrogen bonds. These four hydrogen bonds, situated on both sides of the CONH bond, unite the upper part of the helix with the lower one.